1. Field of the Invention
This invention relates to an improved process for obtaining films and blocks of cellulose acetate, particularly but not exclusively suitable for rapid and preparative analytical electrophoresis and for immuno techniques. More specifically, this invention relates to an improved process for obtaining films and blocks which are suitable for the above mentioned electrophoresis purposes, which consist essentially of cellulose diacetate aqueous gel. This invention also relates to blocks and films of cellulose diacetate aqueous gel obtained by said process.
2. Description of the Prior Art
Cellulose acetate membranes (or films) and processes for their preparation have been known long since. The membrane structure and composition and, consequently, their manufacture methods differ widely, however, depending on the membrane end use. The main fields of cellulose acetate membrane application are waste water purification by the reverse osmosis method and analytical electrophoresis.
It should be clearly understood that a cellulose acetate membrane which is successfully suitable for reverse osmosis purposes is not suitable at all for electrophoresis purposes, and vice versa.
The reverse osmosis membranes (or "semipermeable" membranes) formed from cellulose acetate, posses the unique capability of allowing the passage of pure water at reasonably high throughput rates, while rejecting a large percentage of the water-pollutants. The most accepted and experimentally supported theory for the perm-selectivity of the cellulose acetate reverse osmosis membrane suggests that the membrane is composed of a dense "skin," which is only about 0.25 microns in thickness, supported by a porous structure. This dense skin, which would be responsible for the perm-selectivity of the membrane, is composed of crystalline and amorphous zones. In the crystalline zones, the internal stresses restrict the motion of the cellulose biacetate polymer chains. Because of these crystalline zones, the intermolecular distances in the amorphous zones are much smaller than would be expected if there are no crystalline zones.
When the water molecules contact the membrane skin, they concentrate in the amorphous zones whereby they cross-link by hydrogen bonds the cellulose acetate polymer chains. A "pore" in the skin may therefore be envisioned as a small amorphous zone wherein the crosslinking is complete, leaving no "holes" through which ions, which are relatively larger than water molecules, can pass. Water molecules only will be therefore transported across the skin by what is termed "alignement type diffusion" where hydrogen bonds are broken and restored while a water molecule is driven across the skin by pressure differential. The production of cellulose acetate perm-selective membranes is similar to casting procedure for most solvent cast films, except but two essential features. First, the membrane casting solution contains a suitable agent to control membrane porosity, since--as previously indicated--the pores of the skin have to be small enough to prevent the passage of any molecule but water. Second, the membrane is subjected to an annealing step which is usually carried out at temperatures of from about 70.degree. to 85.degree. C. The purpose of the annealing step is, as disclosed for instance in the British Pat. No. 1,159,218 (issued to Aerojet-General Corporation): "to provide a tight membrane having the ability to pass water and restrain passage of salt. During the annealing process a contraction of the swollen gel structure is accomplished. Prior to annealing, the swollen cellulose ester membrane possesses a primary gel structure which exhibits high water transport and low salt retention. Annealing is a syneresis phenomenon, wherein the primary gel structure is shrunk as evidenced by loss of water from the membrane. Annealing may be achieved by immersion of the swollen primar gel structure in a hot water bath."
As disclosed by Manjikian S. et al. ("Improvement in fabrication techniques for reverse osmosis desalination membranes"--First International Symposium on Water Desalination, Oct. 3-9, 1965 Washington D.C.) aqueous magnesium perchlorate was originally included in the casting solution as one of the steps to control reverse osmosis membrane porosity, which was later substituted by formamide as a replacement for both water and magnesium perchlorate, the range of casting solution studied by the foregoing authors being cellulose acetate 20-30 wt.%, formamide 10-40 wt.%, acetone 35-65 wt.%.
The cellulose acetate films suitable for electrophoresis purposes are extremely different from the reverse osmosis membranes particularly as far as the porosity of those materials is concerned. In fact, far from possessing any superficial skin of the afore-mentioned type which allows the passage of the small water molecules only, a material suitable for electrophoresis purposes must have pores of a relatively large diameter to permit the penetration of large organic molecules to be analysed, as for instance hemoglobin whose molecular weight is many thousand times that of water.
These films are commercially available in the form of thin microporous, dry, re-wettable strips with pore sizes up to a diameter of 5 microns, i.e., with pores which are greater than the molecular dimensions of the substances to be analysed by means of electrophoresis. Films of conventional type are moreover produced with thicknesses comprised between 110 and 200 microns only and therefore they are confined to the micro-electrophoretic analysis of highly concentrated samples.
Although these films are advantageous from some points of view, they give rise to other considerable disadvantages which are summarised hereinafter:
1. During immersion in aqueous baths they may trap air in the micropores.
2. Films exposed to air completely lose the water of impregnation in about 30 minutes at a temperature of 20.degree. C. Moreover after a few moments of exposure to air said films lose water to the point of showing clear white spaces when it is well known that for practical use a large capacity for retaining water is required.
3. The films present different characteristics of solubility in solvents according to the degree of acetylation of the starting material, while easy solubility in solvents miscible with water such as acetone is required, together with easy attack by transparentising solvents such as acetic acid.
4. The relatively limited thickness of the films at present available commercially does not allow large volumes of samples to be deposited and analysed, while for unlimited use of electrophoresis the ability to analyse liquids with small concentrations of components in large volumes is required.
5. The relatively large size of the pores of the films at present available on the market may oppose electrophoretic separation because the components of the substance to be analysed spread to a point that they overlap and become confused during the time of electrophoresis. Moreover losses of substances can arise during post-electrophoresis treatments because of flow from the pores.
6. Films at present available in commerce in the dry state are very fragile.
To control pore diameter up to those dimensions which are required by the electrophoretic intended use and reduce film fragility, which constitutes a major problem in the dry, re-wettable cellulose acetate film production, Ueno et al. (U.S. Pat. No. 3,567,809) have disclosed a method which comprises subjecting a film consisting of a mixture of cellulose diacetate and triacetate to a first drying step at a temperature of from 15.degree. C. to 45.degree. C. and a second drying step at a temperature of from 80.degree. C. to 120.degree. C., wherein a plasticizer of suitable hydrophobicity is used.
Finally, methods for producing films of cellulose ester aqueous gel are also known, which generally comprise dissolving a film-forming cellulose ester and a swelling agent in an organic solvent, casting the solution thus obtained to form a film, evaporating at least portion of the organic solvent from the cast film and immersing the cast film in water to substitute water for the organic solvent. These methods are generally unsatisfactory, because it is not possible to regulate the water content of the aqueous gel. As is well known to any expert in the electrophoretic methods and techniques, because of the Joule effect caused by the electric current flowing through the film, an insufficient content of water present within the gel structure may result in an excessively fast drying of the film, which might ultimately cause inaccuracies in the analysis results. Besides, the water molecules linked by hydrogen bonds to cellulose ester polymer chains, affect the movement of high molecular weight molecules to be analysed.